Analysis of intact phosphoinositides in biological samples

Abstract

It is now apparent that each of the known, naturally occurring polyphosphoinositides, the phosphatidylinositol monophosphates (PtdIns3P, PtdIns4P, PtdIns5P), phosphatidylinositol bisphosphates [PtdIns(3,4)P2, PtdIns(3,5)P2, PtdIns(4,5)P2], and phosphatidylinositol trisphosphate [PtdIns(3,4,5)P3], have distinct roles in regulating many cellular events, including intracellular signaling, migration, and vesicular trafficking. Traditional identification techniques require [32P]inorganic phosphate or [3H]inositol radiolabeling, acidified lipid extraction, deacylation, and ion-exchange head group separation, which are time-consuming and not suitable for samples in which radiolabeling is impractical, thus greatly restricting the study of these lipids in many physiologically relevant systems. Thus, we have developed a novel, high-efficiency, buffered citrate extraction methodology to minimize acid-induced phosphoinositide degradation, together with a high-sensitivity liquid chromatography-mass spectrometry (LC-MS) protocol using an acetonitrile-chloroform-methanol-water-ethylamine gradient with a microbore silica column that enables the identification and quantification of all phosphoinositides in a sample. The liquid chromatograph is sufficient to resolve PtdInsP3 and PtdInsP2 regioisomers; however, the require a of and to are using for the of phosphoinositides in and It is now apparent that each of the known, naturally occurring polyphosphoinositides, the phosphatidylinositol monophosphates (PtdIns3P, PtdIns4P, PtdIns5P), phosphatidylinositol bisphosphates [PtdIns(3,4)P2, PtdIns(3,5)P2, PtdIns(4,5)P2], and phosphatidylinositol trisphosphate [PtdIns(3,4,5)P3], have distinct roles in regulating many cellular events, including intracellular signaling, migration, and vesicular trafficking. Traditional identification techniques require [32P]inorganic phosphate or [3H]inositol radiolabeling, acidified lipid extraction, deacylation, and ion-exchange head group separation, which are time-consuming and not suitable for samples in which radiolabeling is impractical, thus greatly restricting the study of these lipids in many physiologically relevant systems. Thus, we have developed a novel, high-efficiency, buffered citrate extraction methodology to minimize acid-induced phosphoinositide degradation, together with a high-sensitivity liquid chromatography-mass spectrometry (LC-MS) protocol using an acetonitrile-chloroform-methanol-water-ethylamine gradient with a microbore silica column that enables the identification and quantification of all phosphoinositides in a sample. The liquid chromatograph is sufficient to resolve PtdInsP3 and PtdInsP2 regioisomers; however, the require a of and to are using for the of phosphoinositides in and of lipids with and roles group are the which a of cellular events, migration, and The of phosphoinositides in and phosphoinositide in in The and of these are phosphoinositides and phosphoinositide phosphoinositide in and lipid the are phosphatidylinositol the of the which phosphatidylinositol and phosphatidylinositol is the to and in and and the of and the of phosphoinositide and and is now apparent that PtdInsP2 phosphatidylinositol and phosphatidylinositol together with the phosphatidylinositol phosphatidylinositol phosphatidylinositol and phosphatidylinositol have and to to the in the of the a phosphoinositide and in many to and a of in and a of the is in and is to in the or to these lipids have in many and the of the phosphoinositides and of the in and in and to the of these we the to that the have identification is is [3H]inositol or [32P]inorganic phosphate radiolabeling of radiolabeling is not for many in for a sufficient of radiolabeling, the lipids are and to and the head are ion-exchange of these are in in is of to the in these techniques for many in which is all to the of the which in the that the of have and and and which are intracellular that the of phosphatidylinositol phosphate is an to novel, to and resolve of spectrometry identification of and PtdInsP3 have of phosphatidylinositol phosphate in lipid using of phosphoinositides using of and a however, these to resolve the these the to of lipid which to a of is the for the we the of liquid chromatography-mass spectrometry that the of all of the phosphoinositides in a lipid and the techniques to physiologically relevant citrate and of the of in and and in with of and in to a of and with or for the of of for and lipids of the lipids of phosphatidylinositol and in to minimize and in in for to and extraction minimize the for of each of PtdIns4P, PtdIns(3,5)P2, and to in to each for a of and the for to the The a using a The with of and the with the of to the extraction with of The with the the in of and a for of phosphoinositides for to to to and for to a for and or a for of the to the for the in or using a a The and with of and identification in that all of the we have each extraction to of is for the extraction, and of these lipids to is these lipids are in the of of the lipid the of lipid of or lipid in the sample. for including and It that is in the of extraction in for these lipids and with of the that is not all an for the liquid chromatograph all with to minimize are that of in the of of a a the phosphoinositides and is that column are and or lipid extraction of and extraction phosphoinositide the phosphate with cellular and in with however, we and of to these we including with and with however, and in to with the the the the in the with is phosphoinositide and phosphate are to in a of of of of of and the of the the of a we acidified with phosphate for extraction of the the and of the of however, the in not the of the with a of the with of the The with extraction is the of which with the of and of we a extraction each of the the of the polyphosphoinositides, in The the of the and thus lipids the of phosphoinositide including samples with of the the that the not resolve the a liquid chromatograph to a to a for the of phosphoinositides the and of of using liquid and or the of a gradient for of the and silica and in and that and with these and for PtdInsP2 is to these the the in the for the of of to is the for and the of to which to of using liquid and or in phosphate not apparent the of phosphoinositide to phosphate or of phosphoinositide the with that with and and with is in in a of the that that the of the for or to the of silica a of column degradation, to the of of to not greatly the of column degradation, with or in many to column with to resolve the lipid with or The of the silica is for of the lipids the The silica is with and the for the of including the and of PtdIns4P, PtdIns(3,5)P2, and is in to the PtdInsP2 however, with the of the and It not to resolve and for the the and these the and with the in in with of the in each of with lipid to resolve phosphoinositides The silica column a of in the however, the gradient to a to PtdInsP3 with the greatly a samples a using a of for the a for of phosphoinositide that the the with a which that for the The with the the Thus, PtdInsP2 and the the of in a and to resolve the to the that we a of of a that phosphate the and the the the a and and however, a these for with and of and in a of the the the the and the the of The of to is for the of and in a of the a the of in the of a and a of a for the of the of a of and The for the samples with with of the to in which the the of or these and the of and with of these a the and a to the for the The a of and to the to the of the to in an the for a in that sample. with the to and to of each and of of the and the of the and of these the together with or the of the a that is to that for for and samples a a with which the to the of the and PtdInsP3 in and The and PtdInsP2 in the are and not the of these in or of of for in a of and The a of that the of to not to an in to to to and the phosphoinositide in together with and and with a and PtdInsP2 in is of the is to that for and not to The of and in the of not and for a for and of and and of all PtdInsP2 and in the and however, and of to with for the and PtdInsP2 in The with in and with a of phosphoinositides of of of of of and of PtdInsP3 column using the in to for and with samples we to of to a of and phosphoinositides or deacylation, have all to to extraction the of the to extraction and to resolve the regioisomers; of have developed and techniques that these and for the an that and the phosphoinositide including the and in the of a liquid chromatograph to a is sufficient for many including identification and quantification of a is identification of is techniques to the of phosphoinositide with phosphate the and of the we are of not the of the these are with these and of phosphoinositides using extraction have not in with identification and quantification of the head ion-exchange the extraction to of and thus not have with a phosphate which not in with an extraction greatly and with with and of and a however, the of the which we not with that and a to to using of of phosphatidylinositol phosphate spectrometry of a for a in The which is for the is a and thus of is not for which have a and in which of are however, a of are is not an all are with of that the the of phosphatidylinositol phosphate we have for in in which is the is with the the methodology for the a and of all phosphoinositides or and enables which of in the study of these in many of lipids with and roles group are the which a of cellular events, migration, and The of phosphoinositides in and phosphoinositide in in The and of these are phosphoinositides and phosphoinositide phosphoinositide in and lipid the are phosphatidylinositol the of the which phosphatidylinositol and phosphatidylinositol is the to and in and and the of and the of phosphoinositide and and is now apparent that PtdInsP2 phosphatidylinositol and phosphatidylinositol together with the phosphatidylinositol phosphatidylinositol phosphatidylinositol and phosphatidylinositol have and to to the in the of the a phosphoinositide and in many to and a of in and a of the is in and is to in the or to these lipids have in many and the of the phosphoinositides and of the in and in and to the of these we the to that the have identification is is [3H]inositol or [32P]inorganic phosphate radiolabeling of radiolabeling is not for many in for a sufficient of radiolabeling, the lipids are and to and the head are ion-exchange of these are in in is of to the in these techniques for many in which is all to the of the which in the that the of have and and and which are intracellular that the of phosphatidylinositol phosphate Thus, is an to novel, to and resolve of spectrometry identification of and PtdInsP3 have of phosphatidylinositol phosphate in lipid using of phosphoinositides using of and a however, these to resolve the these the to of lipid which to a of is the for the we the of liquid chromatography-mass spectrometry that the of all of the phosphoinositides in a lipid and the techniques to physiologically relevant citrate and of the of in and and in with of and in to a of and with or for the of of for and lipids of the lipids of phosphatidylinositol and in to minimize and in in for to and extraction minimize the for of each of PtdIns4P, PtdIns(3,5)P2, and to in to each for a of and the for to the The a using a The with of and the with the of to the extraction with of The with the the in of and a for of phosphoinositides for to to to and for to a for and or a for of the to the for the in or using a a The and with of citrate and of the of in and and in with of citrate and of the of in and and in with of and in to a of and with or for the of of for and lipids of the lipids of phosphatidylinositol and in in to a of and with or for the of of for and lipids of the lipids of phosphatidylinositol and in to minimize and in in for to and to minimize and in in for to and extraction minimize the for of each of PtdIns4P, PtdIns(3,5)P2, and to in to each for a of and the for to the The a using a The with of and the with the of to the extraction with of The with the the in of and a for minimize the for of each of PtdIns4P, PtdIns(3,5)P2, and to in to each for a of and the for to the The a using a The with of and the with the of to the extraction with of The with the the in of and a for of phosphoinositides for to to to and for to a for and or a for of the to the for the of phosphoinositides for to to to and for to a for and or a for of the to the for the in or using a a The and with of in or using a a The and with of and identification in that all of the we have each extraction to of is for the extraction, and of these lipids to is these lipids are in the of of the lipid the of lipid of or lipid in the sample. for including and It that is in the of extraction in for these lipids and with of the that is not all an for the liquid chromatograph all with to minimize are that of in the of of a a the phosphoinositides and is that column are and or lipid extraction of and extraction phosphoinositide the phosphate with cellular and in with however, we and of to these we including with and with however, and in to with the the the the in the with is phosphoinositide and phosphate are to in a of of of of of and the of the the of a we acidified with phosphate for extraction of the the and of the of however, the in not the of the with a of the with of the The with extraction is the of which with the of and of we a extraction each of the the of the polyphosphoinositides, in The the of the and thus lipids the of phosphoinositide including samples with of the the that the not resolve the a liquid chromatograph to a to a for the of phosphoinositides the and of of using liquid and or the of a gradient for of the and silica and in and that and with these and for PtdInsP2 is to these the the in the for the of of to is the for and the of to which to of using liquid and or in phosphate not apparent the of phosphoinositide to phosphate or of phosphoinositide the with that with and and with is in in a of the that that the of the for or to the of silica a of column degradation, to the of of to not greatly the of column degradation, with or in many to column with to resolve the lipid with or The of the silica is for of the lipids the The silica is with and the for the of including the and of PtdIns4P, PtdIns(3,5)P2, and is in to the PtdInsP2 however, with the of the and It not to resolve and for the the and these the and with the in in with of the in each of with lipid to resolve phosphoinositides The silica column a of in the however, the gradient to a to PtdInsP3 with the greatly a samples a using a of for the a for of phosphoinositide that the the with a which that for the The with the the Thus, PtdInsP2 and the the of in a and to resolve the to the that we a of of a that phosphate the and the the the a and and however, a these for with of the a the of in the of a and a of a for the of the of a of and The for the samples with with of the to in which the the of or these and the of and with of these a the and a to the for the The a of and to the to the of the to in an the for a in that sample. with the to and to of each and of of the and the of the and of these the together with or the of the a that is to that for for and samples a a with which the to the of the and PtdInsP3 in and The and PtdInsP2 in the are and not the of these in or of of for in a of and The a of that the of to not to an in to to to and the phosphoinositide in together with and and with a and PtdInsP2 in is of the is to that for and not to The of and in the of not and for a for and of and and of all PtdInsP2 and in the and however, and of to with for the and PtdInsP2 in The with in and with a of phosphoinositides of of of of of and of PtdInsP3 column using the in to for and with samples we to of and identification in that all of the we have each extraction to of is for the extraction, and of these lipids to is these lipids are in the of of the lipid the of lipid of or lipid in the sample. for including and It that is in the of extraction in for these lipids and with of the that is not all an for the liquid chromatograph all with to minimize are that of in the of of a a the phosphoinositides and is that column are of is for the extraction, and of these lipids to is these lipids are in the of of the lipid the of lipid of or lipid in the sample. for including and It that is in the of extraction in for these lipids and with of the that is not all an for the liquid chromatograph all with to minimize are that of in the of of a a the phosphoinositides and is that column are and or lipid extraction of and extraction phosphoinositide the phosphate with cellular and in with however, we and of to these we including with and with however, and in to with the the the the in the with is phosphoinositide and phosphate are to in a of of of of of and the of the the of a we acidified with phosphate for extraction of the the and of the of however, the in not the of the with a of the with of the The with extraction is the of which with the of and of we a extraction each of the the of the polyphosphoinositides, in The the of the and thus lipids the of phosphoinositide including samples with of the the that the not and or lipid extraction of and extraction phosphoinositide the phosphate with cellular and in with however, we and of to these we including with and with however, and in to with the the the the in the with is phosphoinositide and phosphate are to in a of of of of of and the of the the of a we acidified with phosphate for extraction of the the and of the of however, the in not the of the with a of the with of the The with extraction is the of which with the of and of we a extraction each sample. The of the the of the polyphosphoinositides, in The the of the and thus lipids the of phosphoinositide including samples with of the the that the not resolve the a liquid chromatograph to a to a for the of phosphoinositides the and of of using liquid and or the of a gradient for of the and silica and in and that and with these and for PtdInsP2 is to these the the in the for the of of to is the for and the of to which to of using liquid and or in phosphate not apparent the of phosphoinositide to phosphate or of phosphoinositide the with that with and and with is in in a of the that that the of the for or to the of silica a of column degradation, to the of of to not greatly the of column degradation, with or in many to column with to resolve the lipid with or The of the silica is for of the lipids the The silica is with and the for the of including the and of PtdIns4P, PtdIns(3,5)P2, and is in to the PtdInsP2 however, with the of the and It not to resolve and for the the and these the and with the in in with of the in each of with lipid to resolve phosphoinositides The silica column a of in the however, the gradient to a to PtdInsP3 with the greatly a samples resolve the a liquid chromatograph to a to a for the of phosphoinositides the and of of using liquid and or the of a gradient for of the and silica and in and that and with these and for PtdInsP2 is to these the the in the for the of The of to is the for and the of to which to of using liquid and or in phosphate not apparent the of phosphoinositide to phosphate or of phosphoinositide the with that with and and with is in in a of the that that the of the for or to the of silica a of column degradation, to the of of to not greatly the of column degradation, with or in many to column with to resolve the lipid with or The of the silica is for of the lipids the The silica is with and the The for the of including the and of PtdIns4P, PtdIns(3,5)P2, and is in to the PtdInsP2 however, with the of the and It not to resolve and for the the and these the and with the in in with of the in each of with lipid to resolve phosphoinositides The silica column a of in the however, the gradient to a to PtdInsP3 with the greatly a samples a using a of for the a for of phosphoinositide that the the with a which that for the The with the the Thus, PtdInsP2 and the the of in a and a using a of for the a for of phosphoinositide that the the with a which that for the The with the the Thus, PtdInsP2 and the the of in a and to resolve the to the that we a of of a that phosphate the and the the the a and and however, a these for with of the a the of in the of a and a of a for the of the of a of and The for the samples with with of the to in which the the of or these and the of and with of these a the and a to the for the The a of and to the to the of the to in an the for a in that sample. with the to and to of each for and samples a The to resolve the to the that we a of of a that phosphate the and the the the a and and however, a these for with of the a the of in the of a and a of a for the of the of a of and The for the samples with with of the to in which the the of or these and the of and with of these a the and a to the for the The a of and to the to the of the to in an the for a in that sample. with the to and to of each a with which the to the of the and PtdInsP3 in and The and PtdInsP2 in the are and not the of these in or of of for in a of and The a of that the of to not to an in to to to and PtdInsP2 in is of the is to that for and not to The of and in the of not and for a for and of and and of all PtdInsP2 and in the and however, and of to with for the and PtdInsP2 in The with in and with a of phosphoinositides of of of of of and of PtdInsP3 column using the in to for and with samples we to of a with which the to the of the and PtdInsP3 in and The and PtdInsP2 in the are and not the of these in or of of for in a of and The a of that the of to not to an in to to to The and PtdInsP2 in is of the is to that for and not to The of and in the of not and for a for and of and and of all PtdInsP2 and in the and however, and of to The of of of of of and of PtdInsP3 column using the in to for and with samples we to of to a of and phosphoinositides or deacylation, have all to to extraction the of the to extraction and to resolve the regioisomers; of have developed and techniques that these and for the an that and the phosphoinositide including the and in the of a liquid chromatograph to a is sufficient for many including identification and quantification of a is identification of is techniques to the of phosphoinositide with phosphate the and of the we are of not the of the these are with these and of phosphoinositides using extraction have not in with identification and quantification of the head ion-exchange the extraction to of and thus not have with a phosphate which not in with an extraction greatly and with with and of and a however, the of the which we not with that and a to to using of of phosphatidylinositol phosphate spectrometry of a for a in The which is for the is a and thus of is not for which have a and in which of are however, a of are is not an all are with of that the the of phosphatidylinositol phosphate we have for in in which is the is with the the methodology for the a and of all phosphoinositides or and enables which of in the study of these in many to a of and phosphoinositides or deacylation, have all to to extraction the of the to extraction and to resolve the regioisomers; of have developed and techniques that these and for the an that and the phosphoinositide including the and in the of a liquid chromatograph to a is sufficient for many including identification and quantification of a is identification of is techniques to the of phosphoinositide with phosphate the and of the we are of not the of the these are with these The and of phosphoinositides using extraction have not in with identification and quantification of the head ion-exchange the extraction to of and thus not have with a phosphate which not in with an extraction greatly and with The with and of and a however, the of the which we not with that and a to to using of of phosphatidylinositol phosphate spectrometry of a for a in The which is for the is a and thus The of is not for which have a and in which of are however, a of are is not an all are with of that the the of phosphatidylinositol phosphate we have for in in which is the is with the the methodology for the a and of all phosphoinositides or and enables which of in the study of these in many study the and